Abstract: We use Escherichia coli as a model organism to define the mechanisms of gene regulation in bacteria. Regulation of gene expression is a critical aspect of bacterial pathogenesis, and much of our understanding has come from studies of E. coli. The vast majority of work on gene expression has focused on protein regulators, but recent studies in eukaryotes have identified thousands of non-coding RNAs (ncRNAs), many of which have regulatory functions. We have recently identified over 1,000 novel transcripts in E. coli that initiate within protein-coding genes, on both DNA strands. We have strong evidence that many of these intragenic RNAs (iRNAs) have a regulatory function. Discovery of pervasive transcription of iRNAs fundamentally changes the established view of bacterial transcriptomes. The goal of this proposal is to determine whether pervasive iRNA transcription is an important, widespread, regulatory mechanism in bacteria, analogous to microRNAs in eukaryotes. To achieve this goal we will use a multidisciplinary approach to (i) comprehensively identify iRNAs, (ii) determine the mechanisms of regulation for key selected iRNA examples, (iii) determine the impact of gene regulation by iRNAs on virulence in a pathogenic strain of E. coli. We are ideally prepared to undertake this project due to my expertise and training in bacterial genetics and genomics, and my willingness to challenge long-standing paradigms in the field of bacterial gene regulation. The Wadsworth Center provides an outstanding environment for these studies because of its excellent research into bacteria and gene regulation, and a strong network of core facilities. The work proposed here will transform our understanding of bacterial gene expression and has major implications for the study of bacterial physiology and pathogenesis. Public Health Relevance: RNA has recently been recognized as a key regulator of gene expression. We have identified over 1,000 novel RNAs in the model bacterium, Escherichia coli. We will determine the impact of these RNAs on gene expression.